The present invention relates to detection and purification of antibody fragments. More particularly, the present invention relates to the detection and purification of Fc and Fab fragments of IgG""s using reagents prepared from the B1 domain of bacterial protein G.
Protein-protein interactions play an essential role in many biological processes. Understanding the energetics of such interactions is of great importance because it defines the necessary concentrations of interacting partners, the rates at which these partners are capable of associating, and the relative concentrations of bound and free proteins in a solution. See Stites. W. E. (1997) Chem. Rev. 97:1233-1250. Well studied classes of protein-protein interactions (Jones, S. and Thornton, J. M. (1996) Proc. Natl. Acad. Sci. USA 93:13-20; LeConte, L. et al. (1999) J. Mol. Biol. 285:2177-2198) include hormone receptor binding and activation (Wells, J. A. and deVos, A. M. (1996) Ann. Rev. Biochem. 65:609-634), antibodies with protein antigens (Davies, D. R. and Cohen, G. H. (1996) Proc. Natl. Acad. Sci. USA 93:7-12), enzyme inhibitor complexes (Tsunogae, Y. et al. (1986) J. Biochem. 100:1637-46), and protein oligomerization (Argos, P. (1988) Prot. Eng. 2:101-113).
B1 is one of the domains of Protein G, a member of an important class of proteins which form IgG-binding receptors on the surface of certain staphylococcal and streptococcal strains, as described by Boyle, M. D. P. (1990) Bacterial lmmunoglobulin-Binding Proteins, Academic Press, San Diego and Frick, I-M. et al. (1992) Proc. Nati. Acad. Sci. USA 89:8532-8536). It has been suggested that these proteins allow the pathogenic bacterium to evade the host immune response by coating the invading bacteria with host antibodies (Goward, C. R. et al. (1993) Trends Biochem. Sci. 18:136-140), thereby contributing significantly to the pathogenicity of these bacteria. Furthermore, protein G has found numerous applications in biotechnology as a reagent for affinity purification of antibodies (Stahl, S. et al. (1993) Current Opinion in Immunology 5:272-277), since it binds to IgGs of many different species and subclasses, as disclosed in Stone, G. C. et al. (1989) J. Immunol. 143:565-570 and in Fahnestock et al. (1990) U.S. Pat. No. 4,977,247. Further characterization of the sequence determinants that contribute to IgG binding may lead to new therapeutics for streptococcal infections and novel immunochemical reagents and thus represents a significant need in the art.
Staphylococcal Protein A competitively binds to a similar site on the Fc fragment of human IgG""s as the B1 domain, involving in both cases hFc residues 252-254, 433-435, and 311, as described by Deisenhofer, J. (1981) Biochemistry 20:2361-2370 and Sauer-Eriksson, A. E. et al. (1995) Structure 3:265-278. Whereas the interactions between B1 and hFc are predominantly polar, half of the protein A interactions are polar and half are hydrophobic. These proteins present an example of two distinct folds which have evolved different structural features to achieve binding at very similar sites on the same target molecule.
The B1 domain of Protein G is a 56-residue domain that folds into a four-stranded xcex2-sheet and one xcex1-helix, as shown by NMR and X-ray crystallography. Despite its small size, the B1 domain has two separate IgG-binding sites on its surface, each interacting respectively with specific, independent sites on the Fab or Fc fragments of the antibody. Compared to most other protein-protein interactions, the Fc-binding site on the B1 protein is somewhat atypical. First, it is predominantly polar rather than hydrophobic in character (Stites, W. E. (1997) Chem. Rev. 97:1233-1250); second, the interfacial area is on the lower end of the observed range, xcx9c700 xc3x852 rather than the average 1200 xc3x852 (Jones, S. and Thornton, J. M. (1996) Proc. Nat. Acad. Sci. USA 93:13-20); third, rather than a planar interaction surface typically observed in heterodimers, this interface is formed by a double xe2x80x9cknobs-into-holesxe2x80x9d interaction (Crick. F. H. C. (1952) Nature 170:882-883; Crick, F. H. C. (1953) Acta Crystallographica 6:689-697) in which a knob from the B1 protrudes into a hole in the hFc, and vice versa.
In view of the presence of these atypical elements within the Fc-binding site on the B1 domain of protein G, the characterization of the energetic contributions of each of these elements on the B1 domain for Fc fragments of IgG""s represents a long-felt and significant need in the art. Indeed, the characterization of the energetic contributions of the atypical elements within the Fc-binding site on the B1 protein would facilitate the development of improved reagents and methods for detection and purification of antibody fragments, among other applications. The development of such reagents and methods thus represents an ongoing need in the art.
In accordance with the present invention, an isolated GB1 domain polypeptide which exhibits binding activity for an Fab fragment of an IgG but exhibits substantially no binding activity for an Fc fragment of an IgG is disclosed.
Preferably, the isolated GB1 domain polypeptide of the present invention further comprises a disrupted xe2x80x9cknobs-into-holesxe2x80x9d binding site for a Fc fragment of an IgG. More preferably still, the isolated GB1 domain polypeptide of the present invention further comprises a mutation at a xe2x80x9cknobs-into-holesxe2x80x9d binding site on the GB1 polypeptide for a Fc fragment of an IgG GB1 domain polypeptide, the mutation comprising an amino acid substitution.
In accordance with the present invention, a method for purifying Fc fragments of IgG""s by affinity chromatography is also disclosed. The method comprising the steps of: (a) contacting a sample comprising IgG Fc and Fab fragments with a GB1 polypeptide of the present invention immobilized to a solid phase support to immobilize the IgG Fab fragments to the solid phase support; and (b) collecting the IgG Fc fragment remaining in the sample.
In accordance with the present invention, a method for purifying Fab fragments of IgG""s by affinity chromatography is also disclosed. The method comprising the steps of: (a) contacting a sample comprising IgG Fc and Fab fragments with a GB1 polypeptide of the present invention immobilized to a solid phase support to immobilize the IgG Fab fragments to the solid phase support; (b) collecting the IgG Fc fragment remaining in the sample; and (c) eluting the IgG Fab fragments from the solid phase support to give purified IgG Fab fragments in the eluate.
In accordance with the present invention, a method for detecting IgG, a fragment of an IgG, or combinations thereof, in a fluid sample suspected of containing IgG, a fragment of an IgG, or combinations thereof is also disclosed. The method comprising the steps of: (a) contacting the fluid sample with a binding substance comprising the GB1 polypeptide of claim 1, under conditions favorable to binding of IgG, a fragment of an IgG, or combinations thereof to the binding substance to form a complex therebetween; and (b) detecting the complex by means of a label conjugated to the binding substance or by means of a labeled reagent that specifically binds to the complex subsequent to its formation.
It is thus an object of the present invention to provide novel reagents and methods for the detection and purification of antibody fragments.
It is another object of the present invention to novel reagents and methods for the detection and purification of antibody fragments which provides for the separation of Fc fragments and Fab fragments of an IgG, preferably an IgG of a warm-blooded vertebrate.
It is yet another object of the present invention to novel reagents and methods for the detection and purification of antibody fragments which provides for the separation of Fc fragments and Fab fragments of a human IgG.
It is still another object of the present invention to provide for the characterization of the energetic contributions of elements within the Fc-binding site on the B1 domain of protein G to binding with an Fc fragment of an IgG, preferably an IgG of a warm-blooded vertebrate.